禽4型腺病毒单克隆抗体的制备、鉴定与初步应用

doi:10.11843/j.issn.0366-6964.2025.05.033

Abstract

Abstract:

The aim of this study was to prepare a monoclonal antibody to avian adenovirus type 4 (FAdV-4), to characterize its specific binding site, and to evaluate its value for use in immunohistochemistry (IHC) and immunoprecipitation reactions. Mice were immunized with purified FAdV-4-AH-F41 whole virus, a positive hybridoma cell line was screened and subcloned by indirect enzyme linked immunosorbent assay (ELISA), and the prepared monoclonal antibody was tested for potency. Indirect immunofluorescence assay (IFA) and protein immunoblotting (Western blot) were used to characterize the biological properties of the monoclonal antibodies. pCold TF-Hexon, pET-32a(+)-Fiber1, pCold TF-Penton, pCold TF-Fiber2 prokaryotic expression vectors, and the specific binding sites of the resulting monoclonal antibodies were preliminarily characterized by Western blot, and after the subtypes of the antibodies were detected, they were initially applied to IHC and immunoprecipitation experiments. The results showed that a monoclonal antibody strain with stable secretion of anti-Fiber1 protein was obtained in this experiment, and it was named 5C7. The antibody had a potency of 1∶102 400, and its subtype was IgG-2b, which showed good biological properties. In IHC, obvious lesion characteristics could be observed; in immunoprecipitation experiments, 5C7 could act as a capture antibody and bind specifically to the Fiber1 protein in virus-infected cells. The results suggest that the monoclonal antibody prepared in this experiment by immunizing mice with whole virus particles can specifically recognize the Fiber1 protein and shows good application prospects in IHC and immunoprecipitation reactions, which lays the foundation for the establishment of laboratory pathological diagnosis methods of FAdV-4 and the functional study of Fiber1 protein.

Key words: serotype 4 of fowl adenovirus, monoclonal antibody, antibody identification, fiber 1 protein, IHC, immunoprecipitation

CLC Number:

S852.659.1

ZHANG Yu, CHENG Fanyu, YU Zhaorong, SHAO Ying, WEI Ningbo, CHEN Fangfang, WANG Zhenyu, SONG Xiangjun, TU Jian, QI Kezong*. Preparation, Identification, and Preliminary Application of Monoclonal Antibodies against Fowl Adenovirus Serotype 4[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2364-2371.

Figures/Tables 7

Table 1

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References 30

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包被抗原 Coated antigen	血清稀释度Serum dilution degree
包被抗原 Coated antigen	800	1 600	3 200	6 400	12 800	25 600	51 200	102 400	204 800
P	1.11	1.04	0.73	0.54	0.38	0.27	0.18	0.13	0.10
N	0.07	0.06	0.05	0.06	0.05	0.05	0.06	0.06	0.05
P/N	15.07	17.33	14.16	8.78	8.21	5.78	3.10	2.14	1.89

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Preparation, Identification, and Preliminary Application of Monoclonal Antibodies against Fowl Adenovirus Serotype 4

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